Screening methods for identifying compounds which decrease HIV entry into a cell

ABSTRACT

A peptide has an amino acid sequence having more than 80% homology with the amino acid sequence listed as SEQ ID NO:4. A nucleic acid molecule has more than 80% homology with one of the nucleic acid sequences listed as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. Ligands, anti-ligands, cells vectors relating to the peptide and/or nucleic acid molecule are also used.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/938,703,filed Aug. 24, 2001, now U.S. Pat. No. 6,930,174, issued Aug. 16, 2005,which is a divisional of application Ser. No. 09/626,939, filed Jul. 27,2000, now abandoned, which is a divisional of application Ser. No.08/833,752, filed Apr. 9, 1997, now U.S. Pat. No. 6,448,375, issued Sep.10, 2002, which is a continuation of application Ser. No. 08/810,028,filed Mar. 3, 1997, now abandoned. This application also claims priorityto EP 96870021.1, filed Mar. 1, 1996 and EP 96870102.9, filed Aug. 6,1996. The entire teachings of the above applications are incorporatedherein by reference.

BACKGROUND

1. Field of the Invention

The present invention concerns new peptides and the nucleic acidmolecules encoding said peptides, the vector comprising said nucleicacid molecules, the cells transformed by said vector, inhibitorsdirected against said peptides or said nucleic acid molecules, apharmaceutical composition and a diagnostic and/or dosage devicecomprising said products, and non human transgenic animals expressingthe peptides according to the invention or the nucleic acid moleculesencoding said peptides.

The invention further provides a method for determining ligand binding,detecting expression, screening for drugs binding specifically to saidpeptides and treatments involving the peptides or the nucleic acidmolecules according to the invention.

2. Technological Background of the Art

Chemotactic cytokines, or chemokines, are small signalling proteins thatcan be divided in two subfamilies (CC- and CXC-chemokines) depending onthe relative position of the first two conserved cysteines. Interleukin8 (IL-8) is the most studied of these proteins, but a large number ofchemokines (Regulated on Activation Normal T-cell Expressed and Secreted(RANTES), Monocyte Chemoattractant Protein 1 (MCP-1), MonocyteChemoattractant Protein 2 (MCP-2), Monocyte Chemoattractant Protein 3(MCP-3), Growth-Related gene product α (GROα), Growth-Related geneproduct β (GROβ), Growth-Related gene product γ (GROγ), MacrophageInflammatory Protein 1 α (MIP-1α) and β, etc.) has now been described[4]. Chemokines play fundamental roles in the physiology of acute andchronic inflammatory processes as well as in the pathologicaldysregulations of these processes, by attracting and simulating specificsubsets of leucocytes [32]. RANTES for example is a chemoattractant formonocytes, memory T-cells and eosinophils, and induces the release ofhistamine by basophils. MCP-1, released by smooth muscle cells inarteriosclerotic lesions, is considered as the factor (or one of thefactors) responsible for macrophage attraction and, therefore, for theprogressive aggravation of the lesions [4].

MIP-1α, MIP-1β and RANTES chemokines have recently been described asmajor HIV suppressive factors produced by CD8⁺ T-cells [9].CC-chemokines are also involved in the regulation of human myeloidprogenetor cell proliferation [6, 7].

Recent studies have demonstrated that the actions of CC- andCXC-chemokines are mediated by subfamilies of G protein-coupledreceptors. To date, despite the numerous functions attributed tochemokines and the increasing number of biologically active ligands,only six functional receptors have been identified in human. Tworeceptors for interleukin-8 (IL-8) have been described [20, 29]. One(IL-8RA) binds IL-8 specifically, while the other (IL-8RB) binds IL-8and other CXC-chemokines, like GRO. Among receptors bindingCC-chemokines, a receptor, designated CC-chemokine receptor 1 (CCR1),binds both RANTES and MIP-1α[31], and the CC-chemokine receptor 2 (CCR2)binds MCP-1 and MCP-3 [8, 44, 15]. Two additional CC-chemokine receptorswere cloned recently: the CC-chemokine receptor 3 (CCR3) was found to beactivated by RANTES, MIP-1α and MIP-1β [10]; the CC-chemokine receptor 4(CCR4) responds to MIP-1, RANTES and MCP-1 [37]. In addition to thesesix functional receptors, a number of orphan receptors have been clonedfrom human and other species, that are structurally related to eitherCC- or CXC-chemokine receptors. These include the human BLR1 [13], EBI1[5], LCRI [21], the mouse MIP-1 RL1 and MIP-1 RL2 [17] and the bovinePPR1 [25]. Their respective ligand(s) and function(s) are unknown atpresent.

SUMMARY OF THE INVENTION

The present invention is related to a peptide having at least an aminoacid sequence which presents more than 80%, advantageously more than90%, preferably more than 95%, homology with the amino acid sequence asrepresented in SEQ ID NO. 1.

Preferably, said peptide has also at least an amino acid sequence whichpresents more than 80%, advantageously more than 90%, preferably morethan 95%, homology with the amino acid sequence as represented in SEQ IDNO. 2.

According to another embodiment of the present invention, the peptidehas at least an amino acid sequence which presents more than 80%,advantageously more than 90%, preferably more than 95%, homology withthe amino acid sequence as represented in SEQ ID NO. 3.

The present invention is also related to the amino acid sequence of SEQID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or a portion thereof (representedin the FIG. 1).

A “portion of an amino acid sequence” means one or more amino acidsegments having the same or improved binding properties of the wholepeptide according to the invention. Said portion could be an epitopewhich is specifically binded by a ligand of the peptide which could be aknown “natural ligand” of said peptide, an agonist or an analog of saidligand, or an inhibitor capable of competitively inhibiting the bindingof said ligand to the peptide (including the antagonists of said ligandto the peptide).

Specific examples of said portions of amino acid sequence and theirpreparation process are described in the publication of Rucker J. et al.(Cell, Vol. 87, pp. 437-446 (1996)) incorporated herein by reference.

According to the invention, said portion of the amino acid sequence ofthe peptide according to the invention comprises the N-terminus segmentand the first extracellular loop of the peptide.

Therefore, according to the invention, the amino acid sequence asrepresented in SEQ ID NO. 1 is the common amino acid sequence of SEQ IDNO. 2 and of SEQ ID NO. 3 (see also FIG. 1). Therefore, a firstindustrial application of said amino acid sequence is the identificationof the homology between said amino acid sequence and the screening ofvarious mutants encoding a different amino acid sequence than the onepreviously described, and the identification of various types of patientwhich may present a predisposition or a resistance to the disordersdescribed in the following specification.

Preferably, the peptide according to the invention or a portion thereofis an active CC-chemokine receptor.

Advantageously, the CC-chemokine receptor according to the invention isstimulated by the MIP-1β chemokine at a concentration less or equal to10 nm, and is advantageously also stimulated by the MIP-1α or RANTESchemokines. However, said chemokine receptor is not stimulated by theMCP-1, MCP-2, MCP-3, IL-8 and GROα chemokines.

In addition, the peptide according to the invention or a portion thereofis also a receptor of HIV viruses or a portion of said HIV viruses.

It is meant by “HIV viruses”, HIV-1 or HIV-2 and all the various strainsof HIV viruses which are involved in the development of AIDS. It ismeant by a “a portion of HIV viruses”, any epitope of said viruses whichis able to interact specifically with said receptor. Among said portionsof viruses which may be involved in the interaction with the peptideaccording to the invention, are peptides encoded by the ENV and GAGviruses genes.

Preferably, said portion of HTV viruses is the glycopeptide gp120/160(membrane-bound gp160 or the free gp derived therefrom) or a portionthereof.

It is meant by a “portion of the glycopeptide gp120/160” any epitope,preferably an immuno-dominant epitope, of said glycopeptide which mayinteract specifically with the peptide according to the invention, suchas for instance the V3 loop (third hypervariable domain).

According to another embodiment of the present invention, the peptideaccording to the invention is an inactive CC-chemokine receptor. Anexample of such inactive CC-chemokine receptor is encoded by the aminoacid sequence as represented in SEQ ID NO. 2.

It is meant by an “inactive CC-chemokine receptor” a receptor which isnot stimulated by any known CC-chemokine, especially the MIP-1β, MIP-1αor RANTES chemokines.

The peptide represented in SEQ ID NO. 3 according to the invention is an30 inactive receptor which is not a receptor of HIV viruses or of aportion of said HIV viruses, which means that said inactive receptordoes not allow the entry of said FHV viruses into a cell which presentsat its surface said inactive receptor.

Advantageously, the peptide according to the invention is a humanreceptor.

The present invention concerns also the nucleic acid molecule havingmore than 80%, preferably more than 90%, homology with one of thenucleic acid sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3shown in the FIG. 1.

Preferably, said nucleic acid molecule has at least the nucleic acidsequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 of FIG. 1or a portion thereof.

It is meant by a “portion of said nucleic acid molecule” any nucleicacid sequence of more than 15 nucleotides which could be used in orderto detect and/or reconstitute said nucleic acid molecule or itscomplementary strand. Such portion could be a probe or a primer whichcould be used in genetic amplification using the PCR, LCR, NASBA or CPRtechniques for instance.

The present invention concerns more specifically the nucleic acidmolecules encoding the peptide according to the invention. Said nucleicacid molecules are RNA or DNA molecules such as a cDNA molecule or agenomic DNA molecule.

The present invention is also related to a vector comprising the nucleicacid molecule according to the invention. Preferably, said vector isadapted for expression in a cell and comprises the regulatory elementsnecessary for expressing the amino acid molecule in said celloperatively linked to the nucleic acid sequence according to theinvention as to permit expression thereof.

Preferably, said cell is chosen among the group consisting of bacterialcells, yeast cells, insect cells or mammalian cells. The vectoraccording to the invention is a plasmid, preferably a pcDNA3 plasmid, ora virus, preferably a baculovirus, an adenovirus or a semliki forestvirus.

The present invention concerns also the cell, preferably a mammaliancell, such as a CHO-K1 or a HEK293 cell, transformed by the vectoraccording to the invention. Advantageously, said cell is non neuronal inorigin and is chosen among the group consisting of CHO-K1, HEK293,BHK21, COS-7 cells.

The present invention also concerns the cell (preferably a mammaliancell such as a CHO-K1 cell) transformed by the vector according to theinvention and by another vector encoding a protein enhancing thefunctional response in said cell. Advantageously, said protein is theGα15 or Gα16 (G protein, α subunit). Advantageously, said cell is thecell CHO-K1-pEFIN hCCR5-1/16.

The present invention is also related to a nucleic acid probe comprisinga nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a unique sequence included within thesequence of the nucleic acid molecule according to the invention. Saidnucleic acid probe may be a DNA or an RNA.

The invention concerns also an antisense oligonucleotide having asequence capable of specifically hybridizing to an MRNA moleculeencoding the peptide according to the invention so as to preventtranslation of said MRNA molecule or an antisense oligonucleotide havinga sequence capable of specifically hybridizing to the CDNA moleculeencoding the peptide according to the invention.

Said antisense oligonucleotide may comprise chemical analogs ofnucleotide or substances which inactivate MRNA, or be included in an RNAmolecule endowed with ribozyme activity.

Another aspect of the present invention concerns a ligand or ananti-ligand (preferably an antibody) other than known “natural ligands”,which are chosen among the group consisting of the MIP-1β 13, MIP-1α orRANTES chemokines, HIV viruses or a portion of said HIV viruses, whereinsaid ligand is capable of binding to the receptor according to theinvention and wherein said anti-ligand is capable of (preferablycompetitively) inhibiting the binding of said known “natural ligand” orthe ligand according to the invention to the peptide according to theinvention.

The exclusion in the above identified definition of known chemokines,HIV viruses or a portion of said HIV viruses, does not include variantsof said “natural” viruses or said “natural” portion which may beobtained for instance by genetic engineering and which may mimic theinteraction of said viruses and portion of said viruses to the peptideaccording to the invention.

Advantageously, said antibody is a monoclonal antibody which ispreferably directed to an epitope of the peptide according to theinvention and present on the surface of a cell expressing said peptide.

Preferably, said antibody is produced by the hybridome cellAchCCR5-SAB1A7.

The invention concerns also the pharmaceutical composition comprisingeither an effective amount of the peptide according to the invention (inorder to delude the HIV virus from the natural peptide present at thesurface of a mammalian cell and stop the infection of said mammaliancell by the HIV virus), or an effective amount of the above identifieddescribed ligand and/or anti-ligand, or an effective amount ofoligonucleotide according to the invention, effective to decrease theactivity of said peptide by passing through a cell membrane and bindingspecifically with MRNA encoding the peptide according to the inventionin the cell so as to prevent it translation. The pharmaceuticalcomposition comprises also a pharmaceutically acceptable carrier,preferably capable of passing through said cell membrane.

Preferably, in said pharmaceutical composition, the oligonucleotide iscoupled to a substance, such as a ribozyme, which inactivates MRNAencoding the peptide according to the invention.

Preferably, the pharmaceutically acceptable carrier comprises astructure which binds to a receptor on a cell capable of being taken upby cell after binding to the structure. The structure of thepharmaceutically acceptable carrier in said pharmaceutical compositionis capable of binding to a receptor which is specific for a selectedcell type.

The present invention concerns also a transgenic non human mammaloverexpressing (or expressing ectopically) the nucleic acid moleculeencoding the peptide according to the invention.

The present invention also concerns a transgenic non human mammalcomprising an homologous recombination knockout of the native peptideaccording to the invention.

According to a preferred embodiment of the invention, the transgenic nonhuman mammal whose genome comprises antisense nucleic acid complementaryto the nucleic acid according to the invention is so placed as to betranscripted into antisense MRNA which is complementary to the MRNAencoding the peptide according to the invention and which hybridizes toMRNA encoding said peptide, thereby reducing its translation.Preferably, the transgenic non human mammal according to the inventioncomprises a nucleic acid molecule encoding the peptide according to theinvention and comprises additionally an inducible promoter or a tissuespecific regulatory element.

Preferably, the transgenic non human mammal is a mouse.

The invention relates to a method for determining whether a ligand canbe specifically bound to the peptide according to the invention, whichcomprises contacting a cell transfected with a vector expressing thenucleic acid molecule encoding said peptide with the ligand underconditions permitting binding of ligand to such peptide and detectingthe presence of any such ligand bound specifically to said peptide,thereby determining whether the ligand binds specifically to saidpeptide.

The invention relates to a method for determining whether a ligand canspecifically bind to a peptide according to the invention, whichcomprises preparing a cell extract from cells transfected with a vectorexpressing the nucleic acid molecule encoding said peptide, isolating amembrane fraction from the cell extract, contacting the ligand with themembrane fraction under conditions permitting binding of the ligand tosuch peptide and detecting the presence of any ligand bound to saidpeptide, thereby determining whether the compound is capable ofspecifically binding to said peptide. Preferably, said method is usedwhen the ligand is not previously known.

The invention relates to a method for determining whether a ligand is anagonist of the peptide according to the invention, which comprisescontacting a cell transfected with a vector expressing the nucleic acidmolecule encoding said peptide with the ligand under conditionspermitting the activation of a functional peptide response from the celland detecting by means of a bio-assay, such as a modification in asecond messenger concentration (preferably calcium ions or inositolphosphates such as IP₃) or a modification in the cellular metabolism(preferably determined by the acidification rate of the culture medium),an increase in the peptide activity, thereby determining whether theligand is a peptide agonist.

The invention relates to a method for determining whether a ligand is anagonist of the peptide according to the invention, which comprisespreparing a cell extract from cells transfected with a vector expressingthe nucleic acid molecule encoding said peptide, isolating a membranefraction from the cell extract, contacting the membrane fraction withthe ligand under conditions permitting the activation of a functionalpeptide response and detecting by means of a bio-assay, such as amodification in the production of a second messenger (preferablyinositol phosphates such as IP₃), an increase in the peptide activity,thereby determining whether the ligand is a peptide agonist.

The present invention relates to a method for determining whether aligand is an antagonist of the peptide according to the invention, whichcomprises contacting a cell transfected with a vector expressing thenucleic acid molecule encoding said peptide with the ligand in thepresence of a known peptide agonist, under conditions permitting theactivation of a functional peptide response and detecting by means of abio-assay, such as a modification in second messenger concentration(preferably calcium ions or inositol phosphates such as IP₃) or amodification in the cellular metabolism (preferably determined by theacidification rate of the culture medium), a decrease in the peptideactivity, thereby determining whether the ligand is a peptideantagonist.

The present invention relates to a method for determining whether aligand is an antagonist of the peptide according to the invention, whichcomprises preparing a cell extract from cells transfected with anexpressing the nucleic acid molecule encoding said peptide, isolating amembrane fraction from the cells extract, contacting the membranefraction with the ligand in the presence of a known peptide agonist,under conditions permitting the activation of a functional peptideresponse and detecting by means of a bio-assay, such as a modificationin the production of a second messenger, a decrease in the peptideactivity, thereby determining whether the ligand is a peptideantagonist.

Preferably, the second messenger assay comprises measurement of calciumions or inositol phosphates such as IP₃.

Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as CHO-K1, HEK293, BHK21, COS-7 cells.

In said method, the ligand is not previously known.

The invention is also related to the ligand isolated and detected by anyof the preceding methods.

The present invention concerns also the pharmaceutical composition whichcomprises an effective amount of an agonist or an antagonist of thepeptide according to the invention, effective to reduce the activity ofsaid peptide and a pharmaceutically acceptable carrier.

It is meant by “an agonist or an antagonist of the peptide according tothe invention”, all the agonists or antagonists of the known “naturalligand” of the peptide as above described.

Therefore, the previously described methods may be used for thescreening of drugs to identify drugs which specifically bind to thepeptide according to the invention.

The invention is also related to the drugs isolated and detected by anyof these methods.

The present invention concerns also a pharmaceutical compositioncomprising said drugs and a pharmaceutically acceptable carrier.

The invention is also related to a method of detecting expression of apeptide according to the invention by detecting the presence of MRNAcoding for a peptide, which comprises obtaining total RNA or total MRNAfrom the cell and contacting the RNA or MRNA so obtained with thenucleic acid probe according to the invention under hybridizingconditions and detecting the presence of MRNA hybridized to the probe,thereby detecting the expression of the peptide by the cell.

Said hybridization conditions are stringent conditions.

The present invention concerns also the use of the pharmaceuticalcomposition according to the invention for the treatment and/orprevention of inflammatory diseases, including rheumatoid arthritis,glomerulonephritis, asthma, idiopathic pulmonary fibrosis and psoriasis,viral infections including Human Immunodeficiency Viruses 1 and 2 (HIV-1and 2), cancer including leukaemia, atherosclerosis and/or auto-immunedisorders.

The present invention concerns also a method for diagnosing apredisposition or a resistance to a disorder associated with theactivity of the peptide according to the invention and/or associatedwith infectious agents such as HIV viruses in a subject. Said methodcomprises

-   a) obtaining nucleic acid molecules encoding the peptide according    to the invention from the cells of the subject;-   b) possibly performing a restriction digest of said nucleic acid    molecules with a panel of restriction enzymes;-   c) possibly electrophoretically separating the resulting nucleic    acid fragments on a sized gel;-   d) contacting the resulting gel or the obtained nucleic acid    molecule with a nucleic acid probe labelled with a detectable marker    and capable of specifically hybridizing to said nucleic acid    molecule (said hybridization being made in stringent hybridization    conditions);-   e) detecting labelled bands or the in situ nucleic acid molecules    which have hybridized to the said nucleic acid molecule labelled    with a detectable marker to create a unique band pattern or an in    situ marking specific to the subject;-   f) preparing other nucleic acid molecules encoding the peptide    according to the invention obtained from the cells of other patients    for diagnosis by step a-e; and-   g) comparing the unique band pattern specific to the nucleic acid    molecule of subjects suffering from the disorder from step e and the    nucleic acid molecule obtained for diagnosis from step f to    determine whether the patterns are the same or different and to    diagnose thereby a predisposition or a resistance to the disorder if    the patterns are the same or different.

The present invention is also related to a method for diagnosing apredisposition or a resistance to a disorder associated with theactivity of a specific allele of the peptide according to the inventionor the presence of said peptide at the surface of cells and/orassociated with infectious agents such as HIV viruses present in asubject. Said method comprises:

-   a) obtaining a sample of a body fluid, preferably a blood sample    comprising antigen presenting cells, from a subject;-   b) adding to said sample a ligand and/or an anti-ligand according to    the invention;-   c) detecting the cross-reaction between said ligand and/or said    anti-ligand and the specific peptide according to the invention; and-   d) determining whether the peptide corresponds to a receptor or an    inactive receptor according to the invention and diagnosing thereby    a predisposition or a resistance to the disorder according to the    type of the peptide present in the body fluid of the subject.

The present invention concerns also a diagnostic and/or dosage device,preferably a kit, comprising the peptides, the nucleic acid molecules,the nucleic acid probes, the ligands and/or the anti-ligands accordingto the invention, their portions (such as primers, probes, epitopes, . .. ) or a mixture thereof, being possibly labelled with a detectablemarker.

Said diagnostic and/or dosage device comprises also the reactants forthe detection and/or the dotage of antigens, antibodies or nucleic acidsequences through a method selected from the group consisting of in situhybridization, hybridization or recognition by marked specificantibodies, specially ELISA® (Enzyme Linked Immunosorbent Assay) or RIA®(Radio Immunoassay), methods on filter, on a solid support, in solution,in “sandwich”, on gel, by Dot blot hybridization, by Northern blothybridization, by Southern blot hybridization, by isotopic ornon-isotopic labelling (such as immunofluorescence or biotinylation), bya technique of cold probes, by genetic amplification, particularly PCR,LCR, NASBA or CPR, by a double immunodiffusion, by acounter-immunoelectrophoresis, by haemagglutination and/or a mixturethereof.

A last aspect of the present invention concerns a method of preparingpeptides according to the invention, which comprises

-   a) constructing a vector adapted for expression in a cell which    comprises the regulatory elements necessary for the expression of    nucleic acid molecules in the cell operatively linked to nucleic    acid molecule encoding said peptide so as to permit expression    thereof, wherein the cell is preferably selected from the group    consisting of bacterial cells, yeast cells, insect cells and    mammalian cells;-   b) inserting the vector of step a in a suitable host cell;-   c) incubating the cell of step b under conditions allowing the    expression of the peptide according to the invention;-   d) recovering the peptide so obtained; and-   e) purifying the peptide so recovered, thereby preparing an isolated    peptide according to the invention.

The deposits of micro-organisms AchCCR5-SAB1A7 and CHO-K1-PEFINHCCR5-1/16 were made according to the Budapest Treaty in the BelgiumCoordinated Collection of Microorganisms (BCCM), Laboratorium voorMoleculaire Biologic (LMBP), Universiteit Gent, K. L. Ledeganckstraat35, B-9000 GENT, BELGIUM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1, 1A-2, 1B-1, 1B-2, 1B-3, 1D-1, 1D-2 and 1D-3 shows thenucleic acid and amino acid sequences of the invention. FIGS. 1A-1 and1A-2 show the nucleic acid and amino acid sequence of SEQ ID Nos 1 and4, respectively. FIGS. 1B-1, 1B-2, and 1B-3 show the nucleic acid andamino acid sequence of SEQ ID Nos 2 and 5, respectively. FIG. 1D-1 to1D-3 show the nucleic acid and amino acid sequence of SEQ ID Nos. 3 and6, respectively.

FIGS. 2A and 2B represents the amino acids sequence of the active humanCCR5 chemokine receptor (SEQ ID NO:5) according to the invention alignedwith that of the human CCR1 (hCC-R1) (SEQ ID NO:9), CCR2b (hCC-R2b) (SEQID NO:7), CCR3 (hCC-R3) (SEQ ID NO:8) and CCR4 (hCC-R4) (SEQ ID NO:10)receptors. Amino acids identical with the active CCR5 sequence areboxed.

FIG. 3 shows the chromosomal organization of the human CCR2 and CCR5chemokine receptor genes.

FIGS. 4A, 4B and 4C shows the functional expression of the human activeCCR5 receptor in a CHO-K1 cell line.

FIG. 5 represents the distribution of MRNA encoding the CCR5 receptor ina panel of human cell lines of haematopoietic origin.

FIGS. 6A and 6B represents the structure of the mutant form of humanCCR5 receptor. FIG. 6A shows a diagram of the mutant form of human CCR5receptor (SEQ ID NO:18) situated in a membrane. FIG. 6B shows the wildtype amino acid sequence (CCR5) (SEQ ID NO: 11), and the location of the32 base deletion mutation in the nucleic acid sequence encoding CCR5(SEQ ID NO: 12) and amino acid sequences of ΔCCR5 (SEQ ID NO: 13).

FIGS. 7A and 7B represents the quantification of ENV proteins-mediatedfusion by luciferase assays.

FIG. 8 represents genotyping of individuals by PCR and segregation ofthe CCR5 alleles in CEPH families.

FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G and 9H represents the FACS analysis ofsera anti-CCR5 on a CCR5-CHO cell line according to the invention.

FIG. 10 represents the inhibition of HIV infectivity with anti-CCR5antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. Experimentals

Materials

Recombinant human chemokines, including MCP-1, MIP-1α, MIP-1β, RANTES,IL-8 and GROα were obtained from R & D Systems (London, UK).[¹²⁵I]MIP-1α (specific activity, 2200 Ci/mmol) was obtained from DupontNEN (Brussels, Belgium). Chemokines obtained from R & D Systems werereported by the supplier as >97% pure on SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis) and biologically active ona bioassay specific for each ligand. The lyophilised chemokines weredissolved as a 100 μg/ml solution in a sterile phosphate-buffered saline(PBS) and this stock solution was stored at −20° C. in aliquots.Chemokines were diluted to the working concentration immediately beforeuse. All cell lines used in the present study were obtained from theATCC (Rockville, Md., USA).

Cloning and Sequencing

The mouse MOP020 clone was obtained by low stringency polymerase chainreaction, as described previously [24, 34], using genomic DNA astemplate. A human genomic DNA library (Stratagene, La Jolla, Calif.),constructed in the lambda DASH vector was screened at low stringency[39] with the MOP020 (511 bp) probe. The positive clones were purifiedto homogeneity and analysed by Southern blotting. The restriction map ofthe locus was determined and a relevant XbaI fragment of 4,400 bp wassubcloned in pBluescript SK+ (Stratagene). Sequencing was performed onboth strands after subcloning in M13mp derivatives, using fluorescentprimers and an automated DNA sequencer (Applied Biosystem 370A).Sequence handling and data analysis was carried out using theDNASIS/PROSIS software (Hitachi), and the GCG software package (GeneticsComputer Group, Wisconsin).

Expression in Cell Lines

The entire coding region was amplified by PCR as a 1056 bp fragment,using primers including respectively the BamHI and XbaI recognitionsequences, and cloned after restriction in the corresponding sites ofthe eukaryotic expression vector pcdna3 (Invitrogen, San Diego, Calif.).The resulting construct was verified by sequencing, and transfected inCHO-K1 cells as described [35]. Two days after transfection, selectionfor stably transfected cell lines was initiated by the addition of 400μg/ml G418 (Gibco), and resistant clones were isolated at day 10. CHO-K1cells were cultured using Ham's F12 medium, as previously described [35,11]. The expression of the active CCR5 receptor in the various cellclones was evaluated by measuring the specific transcript level byNorthern blotting, on total RNA prepared from the cells (see below).

Binding Assays

Stably transfected CHO-K1 cells expressing the active CCR5 receptor weregrown to confluence and detached from culture dishes by incubation inphosphate-buffered saline (PBS) supplemented with 1 Mm EDTA. Cells werecollected by low speed centrifugation and counted in a Neubaeur cell.Binding assays were performed in polyethylene minisorp tubes (Nunc) in afinal volume of 200 μl PBS containing 0.2% bovine serum albumin (BSA)and 10⁶ cells, in presence of [¹²⁵I]-MIP-1α. Non specific binding wasdetermined by addition of 10 Nm unlabelled MIP-1α. The concentration oflabelled ligand was 0.4 Nm (around 100,000 cpm per tube). The incubationwas carried out for 2 hours at 4° C., and was stopped by the rapidaddition of 4 ml ice-cold buffer, and immediate collection of cells byvacuum filtration through GF/B glass fiber filters (Whatmann) pre-soakedin 0.5% polyethyleneinimine (Sigma). Filters were washed three timeswith 4 ml ice-cold buffer and counted in a gamma counter.

Biological Activity

The CHO-K1 cell lines stably transfected with the pcdna3/CCR5 constructor wild type CHO-K1 cells (used as controls) were plated onto themembrane of Transwell cell capsules (Molecular Devices), at a density of2.5 10⁵ cells/well in Ham's F12 medium. The next day, the capsules weretransferred in a microphysiometer (Cytosensor, Molecular Devices), andthe cells were allowed to equilibrate for approximately two hours byperfusion of 1 Mm phosphatebuffered (Ph 7.4) RPMI-1640 medium containing0.2% BSA. Cells were then exposed to various chemokines diluted in thesame medium, for a 2 mm duration. Acidification rates were measured atone minute intervals.

Northern Blotting

Total RNA was isolated from transfected CHO-K1 cell lines, from a panelof human cell lines of haematopoietic origin and from a panel of dogtissues, using the RNeasy kit (Qiagen). RNA samples (10 μg per lane)were denatured in presence of glyoxal [26], fractionated on a 1% agarosegel in a 10 Mm phosphate buffer (Ph 7.0), and transferred to nylonmembranes (Pall Biodyne A, Glen Cove, N.Y.) as described [42]. Afterbaking, the blots were prehybridized for 4 h at 42° C. in a solutionconsisting of 50% formamide, 5× Denhardt solution (1× Denhardt: 0.02%Ficoll, 0.02% polyvinylpyrolidone, 0.02% BSA), 5×SSPE (1×SSPE: 0.18 MNaCl, 10 Mm Na phosphate, 1 Mm EDTA Ph 8.3), 0.3% Sodium DodecylSulphate (SDS), 250 μg per ml denatured DNA from herring testes. DNAprobes were (α³²P)-labelled by random priming [14]. Hybridizations werecarried out for 12 h at 42° C. in the same solution containing 10%(wt/vol) dextran sulphate and the heat denatured probe. Filters werewashed up to 0.1×SSMC (1×SSC: 150 Mm NaCl, 15 Mm Na Citrate Ph 7.0),0.1% SDS at 60° C. and autoradiographed at −70° C. using Amersham β-maxfilms.

2. Results and Discussion

Cloning and Structural Analysis

The sequence homology characterising genes encoding G protein-coupledreceptors has allowed the cloning by low stringency polymerase chainreaction (PCR) of new members of this gene family [24, 34]. One of theclones amplified from mouse genomic DNA, named MOP020 presented strongsimilarities with characterised chemokine receptors, sharing 80%identity with the MCP-1 receptor (CCR2) [8], 65% identity with theMIP-1α/RANTES receptor (CCRI) [31], and 51% identity with IL-8 receptors[20, 30]. The clone was used as a probe to screen a human genomiclibrary. A total of 16 lambda phage clones were isolated. It wasinferred from the restriction pattern of each clone and from partialsequence data that all clones were belonging to a single contig in whichtwo different coding sequences were included. One of the codingsequences was identical to the reported CDNA encoding the CCR receptor[8, 44]. A 4.400 pb XbaI fragment of a representative clone containingthe second region of hybridization was subcloned in Pbluescript SK+.Sequencing revealed a novel gene, tentatively named CCR5, sharing 84%identity with the MOP020 probe, suggesting that MOP020 is the mouseortholog of CCR5. MOP020 does not correspond to any of the three mousechemokine receptor genes cloned recently [16], demonstrating theexistence of a fourth murine chemokine receptor.

The sequence of CCR5 revealed a single open reading frame of 352 codonsencoding a protein of 40,600 Da. The sequence surrounding the proposedinitiation codon is in agreement with the consensus as described byKozak [22], since the nucleotide in −3 is a purine. The hydropathyprofile of the deduced amino acid sequence is consistent with theexistence of 7 transmembrane segments. Alignment of the CCR5 amino acidsequence with that of other functionally characterised humanCC-chemokine receptors is represented in FIG. 2. The highest similarityis found with the CCR2 receptor [8] that shares 75.8% identicalresidues. There is also 56.3% identity with the CCR1 receptor [31],58.4% with the CCR3 [10], and 49.1% with the CCR4 [37]. CCR5 representstherefore a new member of the CC-chemokine receptor group [30]. Like therelated CCR1 and IL-8 receptors [20, 29, 31, 16] the coding region ofCCR5 appears as intronless. From our partial sequencing data, the CCR2gene is also devoid of intron in the first two thirds of its codingsequence.

Sequence similarities within the chemokine receptor family are higher inthe transmembrane-spanning domains, and in intracellular loops. As anexample, the identity score between CCR5 and CCR2 goes up to 92% whenconsidering the transmembrane segments only. Lower similarities arefound in the N-terminal extracellular domain, and in the extracellularloops. The N-terminal domain of the IL-8 and CCR2 receptors has beenshown to be essential for interaction with the ligand [19, 18]. Thevariability of this region among CC-chemokine receptors presumablycontributes to the specificity towards the various ligands of thefamily.

A single potential site for N-linked glycosylation was identified in thethird extracellular loop of CCR5 (FIG. 1). No glycosylation site wasfound in the N-terminal domain of the receptor, where most Gprotein-coupled receptors are glycosylated. The other chemokinereceptors CCR1 and CCR2 present such an N-linked glycosylation site intheir N-terminal domain [31, 8]. By contrast, the CCR3 receptor [10]does not display glycosylation sites neither in the N-terminus, nor inextracellular loops. The active CCR5 receptor has four cysteines in itsextracellular segments, and all four are conserved in the other CC- andCXC-chemokine receptors (FIG. 2). The cysteines located in the first andsecond extracellular loops are present in most G protein-coupledreceptors, and are believed to form a disulphide bridge stabilising thereceptor structure [41]. The two other cysteines, in the N-terminalsegment, and in the third extracellular loop could similarly form astabilising bridge specific to the chemokine receptor family. Theintracellular domains of CCR5 do not include potential sites forphosphorylation by protein kinase C (PKC) or protein kinase A. PKCsites, involved in heterologous desensitisation are frequent in thethird intracellular loop and C-terminus of G protein-coupled receptors.CCR1 is also devoid of PKC sites. In contrast, all CC-chemokinereceptors, are rich in serine and threonine residues in the C-terminaldomain. These residues represent potential phosphorylation sites by thefamily of G protein-coupled receptor kinases, and are probably involvedin homologous desensitisation [41]. Five of these S/T residues areperfectly aligned in all five receptors (FIG. 2).

Physical Linkage of the CCR5 and CCR2 Genes

As stated above, the 16 clones isolated with the MOP020 probecorresponded to a single contig containing the CCR5 and CCR2 genes. Theorganisation of this contig was investigated in order to characterisethe physical linkage of the two receptor genes in the human genome. Acombination of restriction mapping, Southern blotting, fragmentsubcloning and partial sequencing allowed to determine the respectiveborders and overlaps of all clones. Out of the 16 clones, 9 turned outto be characterised by a specific restriction map, and theirorganisation is depicted in FIG. 3. Four of these clones (#11, 18, 21,22) contained the CCR2 gene alone, four clones (#7, 13, 15, 16)contained the ChemR13 gene alone and one clone (#9) contains part ofboth coding sequences. The CCR2 and CCR5 genes are organised in tandem,CCR5 being located downstream of CCR2. The distance separating CCR2 andCCR5 open reading frames is 17.5 kb. The chromosomal localisation of thetandem is presently unknown. Other chemokine receptors have however beenlocated in the human genome: the CCR1 gene was localised by fluorescencein situ hybridization to the p21 region of human chromosome 3 [16]. Thetwo IL-8 receptor genes, and theft pseudogene have been shown to beclustered on the human 2q34-q35 region [1].

Functional Expression and Pharmacoloy of the Active CCR5 Receptor

Stable CHO-K1 cell lines expressing the active CCR5 receptor wereestablished and were screened on the basis of the level of CCR5transcripts as determined by Northern blotting. Three clones wereselected and tested for biological responses in a microphysiometer,using various CC- and CXC-chemokines as potential agonists. Wild typeCHO-K1 dells were used as control to ensure that the observed responseswere specific for the transfected receptor, and did not result from theactivation of endogenous receptors. The microphysiometer allows the realtime detection of receptor activation, by measuring the modifications ofcell metabolism resulting from the stimulation of intracellular cascades[33]. Several studies have already demonstrated the potential ofmicrophysiometry in the field of chemokine receptors. Modifications ofmetabolic activity in human monocytes, in response CC-chemokines, weremonitored using this system [43]. Similarly, changes in theacidification rate of THP-1 cells (a human monocytic cell line) inresponse to MCP-1 and MCP-3 have been measured [36]. The estimation ofthe EC₅₀ for both proteins, using this procedure, was in agreement withthe values obtained by monitoring the intracellular calcium in otherstudies [8, 15].

Ligands belonging to the CC- and CXC-chemokine classes were tested onthe CCR5 transfected CHO-K1 cells. Whereas MIP-1α, MIP-1β and RANTESwere found to be potent activators of the new receptor (FIG. 4), theCC-chemokines MCP-1, MCP-2 and MCP-3, and the CXC-chemokines GROα andIL-8 had no effect on the metabolic activity, even at the highestconcentrations tested (30 Nm). The biological activity of one of thechemokines inducing no response on CCR5 (IL-8) could be demonstrated ona CHO-K1 cell line transfected with the IL-8A interleukin receptor(Mollereau et al., 1993): IL-8 produced a 160% increase in metabolicactivity as determined using the microphysiometer. The biologicalactivity of the MCP-2 and MCP-3 preparations as provided by J. Van Dammehave been widely documented [2, 40]. MIP-1α, MIP-1β and RANTES weretested on the wild type CHO-K1 cells, at a 30 Nm-concentration, and noneof them induced a metabolic response. On the CCR5 transfected CHO-K1cell line, all three active ligands (MIP-1α, MIP-1β and RANTES) caused arapid increase in acidification rate, reaching a maximum by the secondor third minute after perfusion of the ligand. The acidification ratereturned to basal level within 10 minutes. The timing of the cellularresponse is similar to that observed for chemokines on their naturalreceptors in human monocytes [43]. When agonists were applied repeatedlyto the same cells, the response was strongly reduced as compared to thefirst stimulation, suggesting the desensitisation of the receptor. Allmeasurements were therefore obtained on the first stimulation of eachcapsule.

The concentration-effect relation was evaluated for the three activeligands in the 0.3 to 30 Nm range (FIGS. 3B and C). The rank order ofpotency was MIP-1α>MIP-1β=RANTES. At 30 Nm concentrations, the effect ofMIP-1α appeared to saturate (at 156% of baseline level) while MIP-1β andRANTES were still in the ascending phase. Higher concentrations ofchemokines could however not be used. The EC50 was estimated around 3 Nmfor MIP-1α. The concentrations necessary for obtaining a biologicalresponse as determined by using the microphysiometer are in the samerange as those measured by intracellular calcium mobilisation for theCCR1 [31], the CCR2A and B [8], and the CCR3 [10] receptors. The ligandspecificity of CCR5 is similar to that reported for CCR3 [10]. CCR3 wasdescribed as the first cloned receptor responding to MIP-1β. However,MIP-1β at 10 Nm elicits a significant effect on the CCR5, while the sameconcentration is without effect on the CCR3 transfected cells [10].These data suggest that CCR5 could be a physiological receptor forMIP-1β.

Binding experiments using [¹²⁵I]-human MIP-1α as ligand did not allow todemonstrate specific binding to CCR53 expressing CHO-K1 cells, using asmuch as 0.4 Nm radioligand and 1 million transfected cells per tube.Failure to obtain binding data could be attributed to a relatively lowaffinity of the receptor for MIP-1α.

Northern Blotting Analysis

Northern blotting performed on a panel of dog tissues did not allow todetect transcripts for CCR5. Given the role of the chemokine receptorfamily in mediating chemoattraction and activation of various classes ofcells involved in inflammatory and immune responses, the probe was alsoused to detect specific transcripts in a panel of human cell lines ofhaematopoietic origin (FIG. 5). The panel included lymphoblastic (Raji)and T lymphoblastic (Jurkat) cell lines, promyeloblastic (KG-1A) andpromyelocytic (HL-60) cell lines, a monocytic (THP-1) cell line, anerythroleukemia (HEL 92.1.7) cell line, a megakaryoblastic (MEG-O1) cellline, and a myelogenous leukaemia (K-562) cell line. Human peripheralblood mononuclear cells (PBMC), including mature monocytes andlymphocytes, were also tested. CCR5 transcripts (4.4 kb) could bedetected only in the KG-1A promyeloblastic cell line, but were not foundin the promyelocytic cell line HL-60, in PBMC, or in any of the othercell lines tested. These results suggest that the active CCR5 receptorcould be expressed in precursors of the granulocytic lineage.CC-chemokines have been reported to stimulate mature granulocytes [27,38, 23, 2]. However, recent data have also demonstrated a role of CC-and CXC-chemokines in the regulation of mouse and human myeloidprogenitor cell proliferation [6, 7].

CCR5 was shown to respond to MIP-1α, MIP-1β and RANTES, the threechemokines identified as the major HIV-suppressive factors produced byCD8⁺ T cells [9], and released in higher amounts by CD4⁺ T lymphocytesfrom uninfected but multiply exposed individuals [51]. CCR5 represents amajor co-receptor for macrophage-tropic (M-tropic) HIV-1 primaryisolates and strains [45, 50]. M-tropic strains predominate during theasymptomatic phase of the disease in infected individuals, and areconsidered as responsible for HIV-1 transmission. Strains adapted forgrowth in transformed T-cell lines (T-tropic strains) use as aco-receptor LESTR (or fusin) [50], an orphan receptor also belonging tothe chemokine receptor family, but not yet characterized functionally[21, 52, 53]. Dual-tropic viruses, which may represent transitionalforms of the virus in late stages of infection [54] are shown to useboth CCR5 and LESTR as coreceptors, as well as the CC-chemokinereceptors CCR2b and CCR3 [47]. The broad spectrum of co-receptor usageof dual-tropic viruses suggests that within infected individuals, thevirus may evolve at least in part from selection by a variety ofco-receptors expressed on different cell types.

Identification of an Inactive ΔCCR5 Receptor

It is known that some individuals remain uninfected despite repeatedexposure to HIV-1 [55, 56, 51]. A proportion of these exposed-uninfectedindividuals results from the relatively low risk of contamination aftera single contact with the virus, but it has been postulated that trulyresistant individuals do exist. In fact, CD4⁺ lymphocytes isolated fromexposed-uninfected individuals are highly resistant to infection byprimary M-tropic, but not T-tropic HIV-1 strains. Also, peripheral bloodmononuclear cells (PBMC) from different donors are not infected equallywith various HIV-1 strains [57-59]. Given the key role played by CCR5 inthe fusion event that mediates infection by M-tropic viruses, it ispostulated that variants of CCR5 could be responsible for the relativeor absolute resistance to HIV-1 infection exhibited by some individuals,and possibly for the variability of disease progression in infectedpatients [62]. The Inventors selected three HJV-1 infected patientsknown to be slow progressors, and four seronegative individuals ascontrols; the full coding region of their CCR5 gene was amplified by PCRand sequenced. Unexpectedly, one of the slow progressors, but also twoof the uninfected controls, exhibited heterozygosity at the CCR5 locusfor a biallelic polymorphism. The frequent allele corresponded to thepublished CCR5 sequence, while the minor one displayed a 32 bp deletionwithin the coding sequence, in a region corresponding to the secondextracellular loop of the receptor (FIG. 6). The FIG. 6 is the structureof the mutant form of human CC-chemokine receptor 5. FIG. 6 a shows theamino acid sequence of the nonfunctional ΔCCR5 protein is represented.The transmembrane organization is given by analogy with the predictedtransmembrane structure of the wild-type CCR5. Amino acids representedin black correspond to unnatural residues resulting from the frame shiftcaused by the deletion. The mutant protein lacks the last threetransmembrane segments of CCR5, as well as the regions involved in Gprotein-coupling. FIG. 6 b shows the nucleotide sequence of the CCR5gene surrounding the deleted region, and translation into the normalreceptor (top) or the truncated mutant (CCR5, bottom). The 10-bp directrepeat is represented in italics. The full size coding region of theCCR5 gene was amplified by PCR, using 5′ TCGAGGATCCAAGATGGATTATCAAGT-3′(SEQ ID NO: 14) and 5′-CTGATCTAGAGCCATGTGCACAACTCT-3′ (SEQ ID NO: 15) asforward and reverse primers' respectively. The PCR products weresequenced on both strands using the same oligonucleotides as primers, aswell as internal primers, and fluorochrome-labelled didcoxynucleotidesas terminators. The sequencing products were run on an Applied Biosystemsequencer, and ambiguous positions were searched along the codingsequence. When the presence of a deletion was suspected from directsequencing, the PCR products were cloned after restriction with BamHIand XbaI endonucleases into pcDNA3. Several clones were sequenced toconfirm the deletion. The deletion was identical in three unrelatedindividuals investigated by sequencing.

Cloning of the PCR product and sequencing of several clones confirmedthe deletion. The deletion causes a frame shift, which is expected toresult in premature termination of translation. The protein encoded bythis mutant allele (Δccr5) therefore lacks the last three transmembranesegments of the receptor. A 10-bp direct repeat flanking the deletedregion (FIG. 6 b) on both sides is expected to have promoted therecombination event leading to the deletion. Numerous mutagenesisstudies performed on various classes of G protein-coupled receptors,including chemokine receptors, makes it clear that such a truncatedprotein is certainly not functional in terms of chemokine-induced signaltransduction: it lacks the third intracellular loop and C-terminalcytoplasmic domains, the two regions involved primarily in G proteincoupling [41]. In order to test whether the truncated protein was ableto function as a HIV-1 co-receptor, the Inventors tested its ability tosupport membrane fusion by both primary M-tropic and dual-tropic virusENV proteins. The recombinant protein was expressed in quail QT6 cellstogether with human CD4. The QT6 cells were then mixed with HeLa cellsexpressing the indicated viral ENV protein and the extent of cell-cellfusion measured using a sensitive and quantitative gene-reporter assay.In contrast to wild-type CCR5, the truncated receptor did not allowfusion with cells expressing the ENV protein from either M-tropic ordual-tropic viruses (FIG. 7). The FIG. 7 represents the quantificationof ENV protein-mediated fusion by luciferase assay. To quantifycell-cell fusion events, Japanese quail QT6 fibrosarcoma cells weretransfected or cotransfected as indicated with the pcdna3 vector(Invitrogen) containing the coding sequence for wild-type CCR5, thetruncated ccr5 mutant, the CCR2b or the Duffy chemokine receptors, orwith the PCDNA3 vector alone. The target cells were also transfectedwith human CD4 expressed from the CMV promoter and the luciferase geneunder the control of the T7 promoter. HeLa effector cells were infected(MOI=10) with vaccinia vectors expressing T7-polymerase (vTF1.1) andeither the JR-FL (vCB28) or 89.6 (vBD3) envelope proteins. Theluciferase activity resulting from cell fusion is expressed as thepercentage of the activity (in relative light units) obtained forwild-type CCR5. All transfections were performed with an identicalquantity of plasmid DNA using pcdna3 as carrier when necessary. Toinitiate fusion, target and effector cells were mixed in 24 well platesat 37° C. in the presence of ara-C and rifampicin, and allowed to fusefor 8 hours. Cells were lysed in 150 μl of reporter lysis buffer(Promega) and assayed for luciferase activity according to themanufacturer's instructions (Promega).

Coexpression of Δccr5 with wild-type CCR5 consistently reduced theefficiency of fusion for both JR-FL and 89.6 envelopes, as compared withCCR5 alone. Whether this in vitro inhibitory effect (not shared by thechemokine receptor Duffy, used as control) also occurs in vivo ispresently not known. Coexpression with the CCR2b receptor [31], which isthe CC-chemokine receptor most closely related to CCR5 but does notpromote fusion by M-tropic HIV-1 strains [48], did not rescue themutation by formation of a hybrid molecule (FIG. 7).

The FIG. 8 represents genotyping of individuals by PCR and segregationof the CCR5 alleles in CEPH families. FIG. 8 a shows autoradiographyillustrating the pattern resulting from PCR amplification and EcoRIcleavage for individuals homozygous for the wild-type CCR5 allele(CCR5/CCR5), the null ΔCCR5 allele (ΔCCR5/ΔCCR5)-, and for heterozygotes(CCR5/ΔCCR5). A 735 bp PCR product is cleaved into a common band of 332bp for both alleles, and into 403 and 371 bp bands for the wild-type andmutant alleles, respectively. FIG. 8 b shows the segregation of the CCR5alleles in two informative families of the CEPH. Half-black and whitesymbols represent heterozygotes and wild-type homozygotes, respectively.For a few individuals in the pedigrees, DNA was not available (ND: notdetermined). PCRs were performed on genomic DNA samples, using5′-CCTGGCTGTCGTCCATGCTG-3′ (SEQ ID NO: 16 and5′-CTGATCTAGAGCCATGTGCACAACTCT-3′ (SEQ ID NO: 17) as forward and reverseprimers respectively. Reaction mixtures consisted in 30 μl of 10 MmTris-HCl buffer Ph 8.0, containing 50 Mm KCl, 0.75 Mm MgCl₂, 0.2 MmdCTP, dGTP and dTTP, 0.1 Mm dATP, 0.5 μCi [a-³²P]-DATP, 0.01% gelatine,5% DMSO, 200 ng target DNA, 60 ng of each of the primers and 1.5 U Taqpolymerase. PCR conditions were: 93° C. for 2 mm 30; 93° C. for 1 min,60° C. for 1 min, 72° C. for 1 min, 30 cycles; 72° C. for 6 min. Afterthe PCR reaction, the samples were incubated for 60 min at 37° C. with10 U EcoRI, and 2 μl of the denatured reaction mixture was applied ontoa denaturing 5% polyacrylamidc gel containing 35% formamide and 5.6 Murea. Bands were detected by autoradiography.

Based on the 14 chromosomes tested in the first experiment, the deletedΔccr5 allele appeared rather frequent in the Caucasian population. Theaccurate frequency was further estimated by testing (FIG. 8 a) a largecohort of Caucasian individuals, including unrelated members of the CEPH(Centre d'Etude des Polymorphismes Humains) families, part of the IRIBHNstaff, and a bank of anonymous DNA samples from healthy individualscollected by the Genetics Department of the Erasme Hospital in Brussels.From a total of more than 700 healthy individuals, the allelefrequencies were found to be 0.908 for the wild-type allele, and 0.092for the mutant allele (Table I). The genotype frequencies observed inthe population were not significantly different from the expectedHardy-Weinberg distribution (CCR5/CCR5: 0.827 vs 0.824; CCR5/Δccr5:0.162 vs 0.167; Δccr5/Δccr5: 0.011 vs 0.008, p>0.999), suggesting thatthe null allele has no drastic effect on fitness. Using two informativeCEPH families, it was confirmed that the wild-type CCR5 gene and itsΔccr5 variant were allelic, and segregated in a normal mendelian fashion(FIG. 8 b). Interestingly, a cohort of 124 DNA samples originating fromCentral Africa (collected from Zaire, Burkina Fasso, Cameroun, Senegaland Benin) and Japan did not reveal a single Δccr5 mutant allele,suggesting that this allele is either absent or very rare in Asian,African black populations (Table I).

The consequences of the existence of a null allele of CCR5 in the normalCaucasian population were then considered in terms of susceptibility toinfection by HIV-1. If, as it is predicted, CCR5 plays a major (notredundant) role in the entry of most primary virus strains into cells,then Δccr5/Δccr5 individuals should be particularly resistant to HIV-1challenge, both in vitro and in vivo. The frequency of the Δccr5/Δccr5genotype should therefore be significantly lower in HIV-1 infectedpatients, and increased in exposed-uninfected individuals. Also, ifheterozygotes have a statistical advantage due to the lower number offunctional receptors on their white blood cells, or to the possibledominant-negative properties of the mutant allele, the frequency ofheterozygotes (and mutant alleles) should be decreased in HIV-infectedpopulations. These hypotheses were tested by genotyping a large numberof seropositive Caucasian individuals (n=645) belonging to cohortsoriginating from various hospitals from Brussels, Liège and Paris (TableI). Indeed, it was found that within this large series, the frequency ofthe null Δccr5 allele was significantly reduced from 0.092 to 0.05 3(p<10⁻⁵). The frequency of heterozygotes was also reduced from 0.162 to0.106 (p<0.001) and not a single Δccr5/Δccr5 individual could be found(p<0.01).

Altogether, functional and statistical data suggest that CCR5 is indeedthe major co-receptor responsible for natural infection by M-tropicHIV-1 strains. Individuals homozygous for the null Δccr5 allele (about1% of the Caucasian population) have apparently a strong resistance toinfection. It is unclear at this point whether resistance to HIV-1 isabsolute or relative, and whether resistance will vary depending on themode of viral contamination. Larger cohorts of seropositive individualswill have to be tested in order to clarify this point. Heterozygoteshave a milder though significant advantage: assuming an equalprobability of contact with HIV, it can be inferred from Table I thatheterozygotes have a 39% reduction in their likeliness of becomingseropositive, as compared to individuals homozygous for the wild-typeCCR5 allele. Both a decrease in functional CCR5 receptor number, and adominant-negative effect of Δccr5 in vivo, comparable to what isobserved in the in vitro experiments (FIG. 7) are possible explanationsfor this relative protection. The mutant allele, which can be regardedas a natural knock-out in human, is not accompanied by an obviousphenotype in homozygous individuals. Nevertheless, the lack of overtphenotype, taken together with the relative protection thatcharacterizes heterozygous subjects, suggests that pharmacologicalagents that selectively block the ability of HIV-1 to utilize CCR5 as acofactor, could be effective in preventing HIV-1 infection, and would bepredicted not be associated with major side effects resulting from CCR5inactivation. These pharmaceutical agents could be used with othercompounds which are able to block other chemokine receptors used asco-receptors by some HIV-primary isolates in order to infect other cells[47]. The prevalence of the null allele in the Caucasian populationraises the question of whether pandemia of HIV (or related viruses usingthe same co-receptor) have contributed during mankind's evolution tostabilize by selection the mutant ccr5 allele at such a high frequency.

Production of Antibodies Anti-CCR5

Antibodies were produced by genetic immunisation. Six week old femalesbalb/c mice were used. DNA coding for the human CCR5 receptor wasinserted in the expression vector pcdna3 under the control of the CMVpromotor and 100 μg DNA was injected in the anterior tibial muscle, fivedays after pre-treatment of this muscle with cardiotoxine (from venom ofNaja Nigricolis). Injections were repeated twice at three weekintervals. Fifteen days after the last injection, blood was taken fromeach animal and sera were tested for the presence of anti-CCR5antibodies.

Test of Sera Using Fluorescence Activated Cell Sorter (FACS)

Sera were tested by fluorescence activated cell sorting usingrecombinant CHO cells expressing the CCR5 receptor. Briefly, cells weredetached using a PBS-EDTA-EGTA solution and incubated into PBS-BSAmedium for 30 minutes at room temperature with 5 μl serum on the basisof 100,000 cells per tube. Cells were then washed and incubated for 30minutes in ice together with anti-mouse antibody labelled withfluorescein. Cells were washed, taken up into 200 μl of a PBS-BSAsolution and fluorescence was analysed by FACS (FACSCAN,Becton-Dickinson). 10,000 cells were counted. Wild type CHO orrecombinant CHO cells expressing the human CCR2b receptor were used ascontrols.

When tested by FACS analysis 2 weeks after the last injection (FIG. 9),all the sera from mice immunised with CCR5 CDNA, clearly recognised thenative receptor expressed on CHO cells (mean of fluorescence=200),without significant cross reaction with control cells expressing CCR2b(mean of fluorescence=20).

Sera were tested on either a CHO cell line expressing high level of CCR5receptor (black histogram) or a CHO cell line expressing CCR2b receptor(white histogram) as negative control. Each serum was testedindividually.

Antibodies Anti-CCR5 and HIV Infectivity

Peripheral blood mononuclear cells (PBMC) from one donor homozygous fromwild type CCR5 gene, were isolated and cultivated 3 days in presence ofPHA.

On day 4, 800 μl of cells (10⁵ cells/ml) were incubated with 8 μl ofsera from mice immunised with CCR5 CDNA, 30 minutes at 37° C. 1 ml ofviral solution (JRCSF HIV strain) is then added and incubated during 2hours. Cells were then washed twice and cultivated during 15 days.

Aliquot of medium is taken at days 0, 4, 7, 10 and 14 and the dosage ofantigen p24 is performed.

14 days after the beginning of the experiment, one serum (serum B0)totally block the production of p24, indicating its ability to block theinfection of the lymphocytes by this HIV strain (FIG. 10). Other serumsalso exhibit a partial or total effect on this infection (serum A2 andB1). All the other sera did not show any effect on this infection.

Production of Monoclonal Antibodies

Mice with the highest title of CCR5 antibodies were selected formonoclonal antibodies production and injected intravenously with 10⁷recombinant CHO-K1 cells expressing human CCR5 receptors. Three dayslater, animals were sacrificed and fusion of splenic cells or cells fromlymph nodes near the site of injection with SP2/0 myeloma cells, wereperformed. Fusion protocol used was that of Galfre et al. (Nature 266,550 (1977)). A selective HAT (hypoxanthine/aminopterin/thymidin) mediumis used to select hybridomas and their supernatants are tested by FACSusing recombinant CHO cells expressing the human CCR5 receptor, as itwas done for the sera. Positives hybridomas are then cloned by limiteddilution. Clones that are shown positive by FACS analyses are thenexpanded and produced in ascites in balb/C mice.

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1. A method for identifying a compound which decreases infectivity of acell by HIV comprising: (a) contacting a cell which expresses apolypeptide comprising the sequence of SEQ ID NO: 5 with a candidatecompound which binds to said polypeptide; (b) contacting said cell withHIV; and (c) measuring infectivity of said cell by said HIV, wherein ifinfectivity is decreased then said candidate compound is identified as acompound which decreased infectivity of a cell by HIV.
 2. The method ofclaim 1, wherein HIV infectivity is decreased by at least two-fold. 3.The method according to claim 1, wherein said infectivity of the cell byHIV is measured by measuring the production of an HIV protein.
 4. Themethod according to claim 3, wherein said HIV protein is p24.
 5. Amethod for identifying a compound which decreases infectivity of a cellby HIV comprising: (a) contacting a polypeptide of SEQ ID NO:5 with acandidate compound and detecting binding of said candidate compound tosaid polypeptide, wherein if said candidate compound binds to saidpolypeptide, then; (b) contacting a cell which expresses a polypeptidecomprising the sequence of SEQ ID NO: 5 with said candidate compound ofstep (a) which binds to said polypeptide; (c) contacting said cell withHIV; and (d) measuring infectivity of said cell by said HIV, wherein ifinfectivity is decreased then said candidate compound is identified as acompound which decreases infectivity of a cell by HIV.
 6. The method ofclaim 5, wherein HIV infectivity is decreased by at least two-fold. 7.The method according to claim 5, wherein said infectivity of the cell byHIV is measured by measuring the production of an HIV protein.
 8. Themethod according to claim 7, wherein said HIV protein is p24.
 9. Amethod for identifying a compound which decreases entry of HIV into acell comprising: (a) contacting a polypeptide of SEQ ID NO:5 with acandidate compound and detecting binding of said candidate compound tosaid polypeptide, wherein if said candidate compound binds to saidpolypeptide, then; (b) contacting a cell which expresses a polypeptidecomprising the sequence of SEQ ID NO: 5 with said candidate compound ofstep (a) which binds to said polypeptide; (c) contacting said cell withHIV; and (d) measuring the entry of said HIV into said cell, wherein ifentry is decreased then said candidate compound is identified as acompound which decreases the entry of HIV into a cell.
 10. The method ofclaim 9, wherein HIV entry is decreased by at least two-fold.
 11. Themethod according to claim 9, wherein said entry of HIV into a cell ismeasured by measuring the production of an HIV protein.
 12. The methodaccording to claim 11, wherein said HIV protein is p24.